Determination of steroids by competitive immunoassay

ABSTRACT

A method and kit for measurement of asteroid by means of a competitive immunoassay, preferably a competitive enzyme immunoassay. The method and kit involve the use of asteroid analogue conjugated to a label. The steroids that are amenable to detection by the method and kit of the present invention include estradiol and progesterone. 
     The method comprises the steps of: 
     a. incubating a mixture of a test sample suspected of containing a given steroid, a solid phase coupled to an antibody specific for that steroid, and a conjugate of an analogue of that steroid to form steroid/antibody complexes and conjugate/antibody complexes on said solid phase; 
     b. separating said solid phase from said mixture; 
     c. measuring the amount of label present in said mixture or in said solid phase; and 
     d. determining the amount of steroid in said sample from the amount of label. 
     The kit comprises a solid phase coupled to an antibody specific for a steroid and a conjugate of an analogue of that steroid.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to detection and measurement of steroids inbiological fluids, and, more particularly, detection and measurement ofsteroids by means of competitive immunoassay.

2. Discussion of the Art

Detection and measurement of steroids in biological fluids is importantfor a variety of reasons. For example, the amount of a particularsteroid in a biological fluid can be used to assist in diagnosing theoccurrence of an endocrinological disorder, to monitor the amount ofhormone required in hormonal replacement therapy, or to assessfertility. Determination of the presence and amount of steroids in abiological fluid can be determined by competitive diagnostic assay.Small molecule, competitive diagnostic assays require a labeledcomponent that can compete with the analyte for available antibodysites. Examples of the labeled component include radioactive tracers,fluorophore/hapten conjugates, and enzyme/hapten conjugates. Typically,the labeled component consists of the analyte or an analogue of theanalyte coupled to a label. The labeled component is typically referredto as a conjugate.

Estradiol (1,3,5(10)-estratrien-3,17α-diol) is an analyte, the detectionand measurement of which is of great importance in the area of fertilitytesting. Estradiol is secreted by the ovary and placenta. It issynthesized by the aromatization of androgens in the thecal andgranulosa cells of the ovary and placenta. The aromatization isstimulated by follitropin (FSH). Estradiol synthesis in turn stimulatesproduction of lutropin (LH) receptors necessary for the synthesis ofandrogen precursors.

Estradiol is important for female sexual differentiation duringgestation, sexual development at the onset of puberty, and regulation ofthe menstrual cycle. The menstrual cycle is the result of a precisecoordination of the functional characteristics of the central nervoussystem, the hypothalamus, the pituitary, the ovary, and the endometrium,which regulate the cyclic release of Gonadotropin Releasing Hormone(GnRH), LH and FSH, and ovarian steroids (estradiol and progesterone).Estradiol is involved in both the stimulation and inhibition of therelease of the gonadotropins, exerting both a positive and a negativefeedback. Early in the follicular phase, ovarian secretion of estradiolfrom the thecal and granulosa cells is modest. During the follicularphase, estradiol stimulates endometrial growth (repairing theendometrium after menses). Toward mid-cycle, LH production increases andresults in the release of the ovum by the rupture of the developedfollicle. After ovulation, estradiol secretion declines slightly. Duringthe luteal phase, estradiol along with progesterone are secreted by thecorpus luteum, stimulating further endometrial growth. If the ovum isnot fertilized, there is a further drop in estradiol and progesterone.This drop in estradiol and progesterone initiates menses.

The measurement of estradiol is important for the evaluation of normalsexual development (menarche), causes of infertility (anovulation,amenorrhea, dysmenorrhea), and menopause. Normal estradiol levels arelowest at menses and during the early follicular phase (25-75 pg/mL).The levels rise in the late follicular phase to a peak of 200-600 pg/mLjust before the LH surge initiates ovulation. As LH peaks, estradiolbegins to decrease before rising again during the luteal phase (100-300pg/mL). If conception does not take place, estradiol falls further toits lowest levels, thus initiating menses. If conception occurs,estradiol levels continue to rise, reaching levels of 1-5 ng/mL duringthe first trimester, 5-15 ng/mL during the second trimester, and 10-40ng/mL during the third trimester. During menopause, estradiol levelsremain low.

There are various-methods for measuring estradiol levels in serum.However, many of these methods utilize radioactive elements as labelsand suffer from several disadvantages. Several of these methods aredescribed in U.S. Pat. No. 5,342,760 (column 1, line 54 through column3, line 30) and are incorporated herein by reference. U.S. Pat. No.5,342,760 discloses and claims a useful method for determination ofestradiol by competitive immunoassay. However, the method claimed inthis patent is only effective with a limited number of antibodies.

Progesterone (4-pregnen-3,20-dione) is an analyte, the detection andmeasurement of which is of great importance in the area of assessing theoccurrence of ovulation, conception, the risk of abortion, or ectopicpregnancy.

In the mitochondria, cholesterol is first converted to pregnenolone viaa cytochrome P-450 enzyme-dependent side chain cleavage followed byhydroxylation. Pregnenolone is then converted to progesterone in areaction catalyzed by 3β-hydroxysteroid dehydrogenase and isomeraseenzymes (3β-HSD). Progesterone is produced primarily by the corpusluteum of the ovary in normally menstruating women and to a lesserextent by the adrenal cortex. At approximately the sixth week ofpregnancy, the placenta becomes the major producer of progesterone. Inthe circulation of blood, approximately 97-98% of the progesterone isbound to albumin or Cortisol Binding Protein. Progesterone ismetabolized, primarily in the liver, to pregnanediol and its watersoluble sulfate and glucuronide derivatives and excreted in the urine.

The major functions of progesterone are in the preparation of the uterusfor implantation and maintaining pregnancy. During the follicular phase,progesterone levels remain low (0.2-1.5 ng/mL). Following the LH surgeand ovulation, luteal cells in the ruptured follicle produceprogesterone in response to LH. During the luteal phase, progesteronerises rapidly to a maximum of 10-20 ng/mL at the fifth to seventh dayfollowing ovulation. If pregnancy does not occur, progesterone levelsdecrease during the last four days of the menstrual cycle due to theregression of the corpus luteum.

If conception occurs, the levels of progesterone are maintained atmid-luteal levels by the corpus luteum until about the sixth week. Atthat time the placenta becomes the main source of progesterone andlevels rise from approximately 10-50 ng/mL in the first trimester to50-280 ng/mL in the third trimester.

Serum progesterone is a reliable indicator of either natural or inducedovulation because of its rapid rise following ovulation. Disorders ofovulation, including anovulation, are relatively frequent and areresponsible for infertility in approximately 15 to 20% of patients.Progesterone levels are abnormally low in these patients during themid-luteal phase.

Luteal phase deficiency is a reproductive disorder associated withinfertility and spontaneous abortion. It is thought to occur in 10% ofinfertile women. It is believed that infertility and pregnancy wastageassociated with this disorder are caused by inadequate maturation anddevelopment of the endometrium. The failure of the endometrium isthought to be attributable to insufficient progesterone production bythe corpus luteum. Serum progesterone levels in the luteal phase arelower than normal in women with luteal phase deficiency.

Measurement of progesterone in the first ten weeks of gestation has beenshown to be a reliable predictor and an effective tool for the diagnosisand treatment of patients with threatened abortion and ectopicpregnancy. Suppressed progesterone levels (10-15 ng/mL) in the presenceof detectable amounts of human chroionic gonadotropin (hCG) is highlysuggestive of threatened abortion or ectopic pregnancy, regardless ofgestational age.

Typical physiological levels (ng/mL) are as follows.

    ______________________________________                                        Women:                                                                        Normal Cycling;                                                               Follicular            0.5                                                     Ovulatory             0.5-1.5                                                 Luteal                4.0-20.0                                                Other;                                                                        Prepubertal           0.2-0.5                                                 Postmenopausal        0.5                                                     Pregnancy             40-200                                                  Men:                                                                          Prepubertal           0.25                                                    Adult                 0.25                                                    ______________________________________                                    

It has been found to be difficult to obtain antibodies that have theappropriate affinity for the analyte relative to the labeled componentalong with the specificity to allow them to be used effectively in acompetitive immunoassay for steroids, such as, for example, estradioland progesterone. The sources of antibodies are limited in number,thereby resulting in either unavailability or excessively high cost. Theuse of any but a limited number of antibodies in a competitive assayresults in an inadequate dose response, which results in inferiorsensitivity, inferior precision, or both of the foregoing. In addition,even when antibodies that demonstrate an appropriate affinity for ananalyte can be developed, many of these antibodies may demonstrate theundesirable property of high cross-reactivity to structurally-similarsteroids. It would be desirable to provide a competitive immunoassayformat capable of detecting levels of estradiol below 50 pg/mL andlevels of progesterone below 1 ng/mL, concentrations which areclinically useful but difficult to measure.

The probability that a particular anti-steroid antibody/labeledcomponent pair will be useful to prepare a sensitive assay for a givensteroid can be assessed by knowledge of the dose response curve. Thedose response curve for asteroid assay is a plot of the ratio of therate of production of response in the presence of steroid analyte to therate of production of response in the absence of steroid analyte as afunction of the concentration of the steroid analyte. The dose responsecurve for a given steroid assay is unique for each antibody/labeledcomponent combination used and is modulated by the competition betweenantibody analyte and labeled component. Consequently, for any particularanti-steroid antibody/labeled component/steroid analyte combination, arelatively steep dose response curve indicates that the particularcombination will be more likely to provide a clinically useful assay.

SUMMARY OF THE INVENTION

This invention provides a method and kit for measurement of a steroid bymeans of a competitive immunoassay, preferably a competitive enzymeimmunoassay. The method and kit involve the use of asteroid analogueconjugated to a label. The steroids that are amenable to detection bythe method and kit of the present invention include estradiol andprogesterone.

The method comprises the steps of:

a. incubating a mixture of a test sample suspected of containing a givensteroid, a solid phase coupled to an antibody specific for that steroid,and a conjugate of an analogue of that steroid to form steroid/antibodycomplexes and conjugate/antibody complexes on said solid phase;

b. separating said solid phase from said mixture;

c. measuring the amount of label present in said mixture or in saidsolid phase; and

d. determining the amount of steroid in said sample from the amount oflabel.

The kit comprises a solid phase coupled to an antibody specific for asteroid and a conjugate of an analogue of that steroid.

The steroid analogues that are preferred for use in the presentinvention can be represented by the following structural formulae:##STR1##

where R¹ represents a member selected from the group consisting of OH,H, an alkyl group, preferably having 1 to 3 carbon atoms, an alkenylgroup, preferably having 2 to 3 carbon atoms, an alkynyl group,preferably having 2 to 3 carbon atoms, and an alkoxy group, preferablyhaving 1 to 3 carbon atoms;

R² represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R³ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, and an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R⁴ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, and an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R⁵ represents an alkoxy group; preferably having 1 to 3 carbon atoms, orOH;

L represents a label group; provided that the carbon atom at position 16does not contain two substituents that are attached to the carbon atomat position 16 by an oxygen atom, and further provided that the carbonatom at position 17 does not contain two substituents that are attachedto the carbon atom at position 17 by an oxygen atom. ##STR2##

where R⁶ represents a member selected from the group consisting of OH,H, an alkyl group, preferably having 1 to 3 carbon atoms, an alkenylgroup, preferably having 2 to 3 carbon atoms, an alkynyl group,preferably having 2 to 3 carbon atoms, and an alkoxy group, preferablyhaving 1 to 3 carbon atoms;

R⁷ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R⁸ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R⁹ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

L represents a label group; provided that the carbon atom at position 16does not contain two substituents that are attached to the carbon atomat position 16 by an oxygen atom, and further provided that the carbonatom at position 17 does not contain two substituents that are attachedto the carbon atom at position 17 by an oxygen atom. ##STR3##

where R¹⁰ represents a member selected from the group consisting of OH,H, an alkyl group, preferably having 1 to 3 carbon atoms, an alkenylgroup, preferably having 2 to 3 carbon atoms, an alkynyl group,preferably having 2 to 3 carbon atoms, and an alkoxy group, preferablyhaving 1 to 3 carbon atoms;

R¹¹ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R¹² represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R¹³ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3carbon atoms, and an alkoxy group, preferably having 1 to 3carbon atoms;

L represents a label group; provided that the carbon atom at position 16does not contain two substituents that are attached to the carbon atomat position 16 by an oxygen atom, and further provided that the carbonatom at position 17 does not contain two substituents that are attachedto the carbon atom at position 17 by an oxygen atom.

In the Formulae 1a, 1b, and 1c, the label group (L) is represented asbeing attached to a specific ring or to a specific radical. However, itis to be understood that the label group can be attached to the compoundat any position where attachment is possible. Positions of possibleattachment are well known to those of ordinary skill in the art.

The preferred substituents for Formula la include H for R¹, OH for R², Hfor R³, H for R⁴, and OH for R⁵ or OH for R¹, --C.tbd.CH for R², H forR³, H for R⁴, and OH for R⁵ ; the preferred substituents for Formula 1binclude --CH(OH)CH₃ for R⁶, H for R⁷, H for R⁸, and H for R⁹ ; thepreferred substituents for Formula 1c include OH for R¹⁰, H for R¹¹, Hfor R¹², and H for R¹³.

The primary benefit of the present invention is that the performance ofa given antibody can be improved over that obtainable when it is used ina conventional assay format, i.e., when the steroid moiety of theconjugate is of the same chemical structure as the analyte. Heretoforeunacceptable antibodies, i.e., antibodies that provide inadequate doseresponse curves, can now perform satisfactorily in a competitiveimmunoassay where an analogue of the analyte is used in the conjugate,where either the substituents of the steroid moiety of the conjugate aredifferent or the position of the label is different or both thesubstituents of the steroid moiety of the conjugate and the position ofthe label are different.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the dose response curves of variousestrogen-alkaline phosphatase conjugates with anti-estradiol antibody.

FIG. 2 is a graph illustrating the dose response curves of varioussteroid-alkaline phosphatase conjugates with anti-estradiol antibody.

FIG. 3 is a graph illustrating the dose response curves of variousestrogen-alkaline phosphatase conjugates with anti-estradiol antibody.

FIG. 4 is a graph illustrating the dose response curves of variousestrogen-alkaline phosphatase conjugates with anti-estradiol antibody.

FIG. 5 is a graph illustrating the dose response curves of variousestrogen-alkaline phosphatase conjugates with anti-estradiol antibody.

FIG. 6 is a graph illustrating the dose response curves of varioussteroid-alkaline phosphatase conjugates with anti-progesterone antibody.

DETAILED DESCRIPTION

The method of the present invention comprises the steps of

a. incubating a mixture of a test sample suspected of containing a givensteroid, a solid phase coupled to an antibody specific for that steroid,and a conjugate of an analogue of that steroid to form steroid/antibodycomplexes and conjugate/antibody complexes on said solid phase;

b. separating said solid phase from said mixture;

c. measuring the amount of label present in said mixture or in saidsolid phase; and

d. determining the amount of steroid in said sample from the amount oflabel.

The kit of the present invention comprises a solid phase coupled to anantibody specific for asteroid and a conjugate of an analogue of thatsteroid.

As used herein, an "analogue" of a given analyte, i.e., a steroid, is achemical compound that is structurally similar to the analyte, butdiffers from the analyte with respect to at least one of the following:

(1) the orientation of at least one substituent (e.g., α-OH for β-OH);

(2) the identity of a given substituent (e.g., --C.tbd.CH for --H); or

(3) the degree of unsaturation of a ring of the steroid backbone (e.g.,a benzene, cyclohexene, or cyclohexane ring for the A ring of thesteroid).

The A, B, C, and D rings of the cyclopentanophenanthrene ring, whichforms the backbone of the steroid, are labeled in FIG. 10.22 of Textbookof Biochemistry with Clinical Correlations, Second Edition, edited byThomas M. Devlin, John Wiley & Sons, Inc. (1986), page 402, incorporatedherein by reference.

In the case of the analyte estradiol, representative examples ofestradiol analogues include, but are not limited to, those compoundswherein at least one of the following modifications has been made:

(a) β-OH at position 17 is replaced by α-OH;

α-H at position 17 is replaced by the ethynyl group (--C.tbd.CH);

(c) α-H at position 16 is replaced by α-OH; or

(d) the degree of unsaturation in the A ring is changed to provide a3-keto-4-ene, a methyl group is added to the junction of the A ring andthe B ring at position 10, and the label is attached to theaforementioned methyl group at position 19.

However, in the case of the estradiol analogues, at position 17, thesubstituents OH and H cannot be replaced by any substituent attached tothe carbon atom at position 17 by a double bond (e.g., ═O or ═N).

In the case of the analyte progesterone, representative examples ofprogesterone analogues include, but are not limited to, those compoundswherein at least one of the following modifications has been made:

(a) ═O at position 20 is replaced by both β-H and α-OH;

(b) α-H at position 17 is replaced by α-OH; or

(c) the degree of unsaturation in the A ring is changed to provide a3-hydroxy substituted triene, the methyl group is removed from thejunction of the A ring and the B ring at position 10, the β-COCH₃ atposition 17 is replaced by β-H, and the α-H at position 17 is replacedby α-OH.

With respect to the substituent(s) on the estradiol analogue at position17, the β-OH substituent of the estradiol compound can be replaced by anα-OH substituent. However, the carbon atom at position 17 of theestradiol analogue does not contain two substituents that are attachedto the carbon atom at position 17 by an oxygen atom. For example, thecarbon atom at position 17 does not have two --OH substituents. Thesubstituent(s) at position 17 can differ from the --OH substituent ofthe estradiol compound. The substituent(s) at position 17 of theestradiol analogue can be an alkyl group, preferably having from 1 to 3carbon atoms, an alkenyl group, preferably having from 2 to 3 carbonatoms, an alkynyl group, preferably having from 2 to 3 carbon atoms, andalkoxy group, preferably having from 1 to 3 carbon atoms. The alkylgroup, the alkenyl group, the alkynyl group, or the alkoxy group can beunsubstituted or substituted. However, the substituent attached to thecarbon atom at position 17 is not a group that is attached to the carbonatom at position 17 by a double bond. In other words, the substituent atposition 17 is not ═O, ═N--, or the like. With respect to thesubstituent(s) on the estradiol analogue at position 16, thesubstituent(s) can be the same as those substituent(s) that can belocated at position 17. The prohibitions for the substituent(s) atposition 16 are the same as those for the substituent(s) at position 17.The precise nature of the substituents on the aforementioned alkyl,alkenyl, alkynyl, and alkoxy groups is not critical. However, they mustnot adversely affect the immunoassay in which the analyte analogue willbe employed. Representative examples of such substituents include, butare not limited to, the hydroxyl group and alkyl groups.

As used herein, the expression "label group" means a group attached toan antibody or an analyte or an analyte analogue to render the reactionbetween the antibody and the analyte or analyte analogue detectable.Representative examples of labels include enzymes, radioactive labels,fluorescein, and chemicals that produce light. A label is any substancethat can be attached to asteroid or a derivative of asteroid and that iscapable of producing a signal that is detectable by visual orinstrumental means. Various labels suitable for use in this inventioninclude catalysts, enzymes, liposomes, and other vesicles containingsignal producing substances such as chromogens, catalysts, fluorescentcompounds, chemiluminescent compounds, enzymes, and the like. A numberof enzymes suitable for use as labels are disclosed in U.S. Pat. No.4,275,149, incorporated herein by reference. Such enzymes includeglucosidases, galactosidases, phosphatases and peroxidases, such asalkaline phosphatase and horseradish peroxidase, which are used inconjunction with enzyme substrates, such as fluoresceindi(galactopyranoside), nitro blue tetrazolium,3,5',5,5'-tetranitrobenzidine, 4-methoxy-1-naphthol,4-chloro-1-naphthol, 4-methylumbelliferyl phosphate,5-bromo-4-chloro-3-indolyl phosphate, chemiluminescent enzymesubstrates, such as the dioxetanes described in WO 88100694 and EP0-254-051-A2, and derivatives and analogues thereof. Preferably, thelabel is an enzyme and most preferably the enzyme is alkalinephosphatase. This invention involves a method and a kit for detectingand measuring a steroid in biological fluids.

The term "test sample", as used herein, refers to a material suspectedof containing the analyte. The test sample can be used directly asobtained from the source or following a pretreatment to modify thecharacter of the sample. The test sample can be derived from anybiological source, such as a physiological fluid, such as, for example,blood, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine,milk, ascites fluid, synovial fluid, peritoneal fluid, amniotic fluid,and the like. The test sample can be pretreated prior to use, such aspreparing plasma from blood, diluting viscous fluids, and the like.Methods of treatment can involve filtration, distillation, extraction,concentration, inactivation of interfering components, the addition ofreagents, and the like. Other liquid samples besides physiologicalfluids can be used, such as water, food products, and the like, for theperformance of environmental or food production assays. In addition, asolid material suspected of containing the analyte can be used as thetest sample. In some instances it may be beneficial to modify a solidtest sample to form a liquid medium or to release the analyte.

Steroids that have been found to be particularly useful as analytesinclude, but are not limited to, estradiol, progesterone, testosterone,and derivatives of the foregoing.

Compounds that have been found to be particularly suitable for use instep (a) can be analogues of estradiol, analogues of progesterone, andanalogues of testosterone.

The steroid analogues that are preferred for use in the presentinvention can be represented by the following structural formulae:##STR4##

where R¹ represents a member selected from the group consisting of OH,H, an alkyl group, preferably having 1 to 3 carbon atoms, an alkenylgroup, preferably having 2 to 3 carbon atoms, an alkynyl group,preferably having 2 to 3 carbon atoms, and an alkoxy group, preferablyhaving 1 to 3 carbon atoms;

R² represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R³ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, and an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R⁴ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, and an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R⁵ represents an alkoxy group, preferably having 1 to 3 carbon atoms, orOH;

L represents a label group; provided that the carbon atom at position 16does not contain two substituents that are attached to the carbon atomat position 16 by an oxygen atom, and further provided that the carbonatom at position 17 does not contain two substituents that are attachedto the carbon atom at position 17 by an oxygen atom. ##STR5##

where R⁶ represents a member selected from the group consisting of OH,H, an alkyl group, preferably having 1 to 3 carbon atoms, an alkenylgroup, preferably-having 2 to 3 carbon atoms, an alkynyl group,preferably having 2 to 3 carbon atoms, and an alkoxy group, preferablyhaving 1 to 3 carbon atoms;

R⁷ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R⁸ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R⁹ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

L represents a label group; provided that the carbon atom at position 16does not contain two substituents that are attached to the carbon atomat position 16 by an Oxygen atom, and further provided that the carbonatom at position 17 does not contain two substituents that are attachedto the carbon atom at position 17 by an oxygen atom. ##STR6##

where R¹⁰ represents a member selected from the group consisting of OH,H, an alkyl group, preferably having 1 to 3 carbon atoms, an alkenylgroup, preferably having 2 to 3 carbon atoms, an alkynyl group,preferably having 2 to 3 carbon atoms, and an alkoxy group; preferablyhaving 1 to 3 carbon atoms;

R¹¹ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R¹² represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

R¹³ represents a member selected from the group consisting of OH, H, analkyl group, preferably having 1 to 3 carbon atoms, an alkenyl group,preferably having 2 to 3 carbon atoms, an alkynyl group, preferablyhaving 2 to 3 carbon atoms, and an alkoxy group, preferably having 1 to3 carbon atoms;

L represents a label group; provided that the carbon atom at position 16does not contain two substituents that are attached to the carbon atomat position 16 by an oxygen atom, and further provided that the carbonatom at position 17 does not contain two substituents that are attachedto the carbon atom at position 17 by an oxygen atom.

Representative examples of analogues of estradiol suitable for use inthe method and kit of this invention can be represented by the followingstructural formulae: ##STR7##

Representative examples of analogues of progesterone suitable for use inthe method and kit of this invention can be represented by the followingstructural formulae: ##STR8##

In general, the substituents most preferred for the analogues ofestradiol are β-OH and α-C.tbd.CH at position 17, β-H and α-H atposition 16, and --OH at position 3, or β-OH and α-H at position 17, β-Hand α-OH at position 16, and --OH at position 3, or β-H and α-OH atposition 17, β-H and α-H at position 16, and --OH at position 3. Thesubstituents most preferred for the analogues of progesterone areβ-CH(OH)CH3 and α-H at position 17 and α-H and β-H at position 16 orβ-C(O)CH3 and a-OH at position 17 and α-H and β-H at position 16.

The analogues can be prepared by procedures that are well known to oneof ordinary skill in the art. The conjugate is preferably prepared bycoupling the label to the conjugate precursor. Conjugate precursors arecommercially available. Many analogues, such as, for example, the sixrepresentative examples shown above, are commercially available.

Steroid analytes such as estradiol, progesterone, testosterone,prednisone, and cortisone can be detected and measured by commonimmunoassay formats. However, for the sake of brevity, only theestradiol immunoassay techniques will be described in detail. However,one of ordinary skill in the art will appreciate that progesterone,testosterone, prednisone, and cortisone, and other steroids, bothnaturally occurring and synthetic, can be detected and measured bytechniques that are substantially similar to the techniques that willnow be described in detail.

Binding members specific for estradiol include estradiol-specificbinding proteins, such as monoclonal and polyclonal antibodies and otherestradiol specific synthetic or recombinant proteins that specificallybind estradiol. For example, it is well known by those skilled in theart that monoclonal and polyclonal antibodies that specifically bind tosteroids such as estradiol can be produced. When an immunogen comprisingestradiol or a derivative of estradiol coupled to a carrier protein(e.g.; albumin), typically by a covalent bond, is injected into ananimal, the animal's immune system will produce polyclonal antibodiesthat specifically bind to estradiol. General methods for the preparationof monoclonal antibodies to analytes using mice or rats are well knownto those skilled in the art. More recently methods for preparing analytespecific synthetic and recombinant proteins have been reported, and thesame methods can be readily adapted to the preparation of estradiolspecific synthetic and recombinant proteins that are useful in thisinvention.

Preferably, estradiol is measured by using a solid phase having abinding member specific for estradiol bound thereto. An example of asolid phase is a microparticle. The solid phase and the test sample areseparated so that the amount of steroid conjugate bound to the solidphase or the amount of steroid conjugate remaining in solution can bedetermined. Preferably, the amount of steroid conjugate bound to thesolid phase or the amount of steroid conjugate remaining in solution isdetermined by enzyme immunoassay, wherein an enzyme is used as the labelin the conjugate. A material referred to as an enzyme substrate can beconverted into a fluorescent compound, such as 4-methylumbelliferone, byan appropriate enzyme. The rate at which the fluorescent compound isformed is an indication of the quantity of enzyme present in thereaction mixture. When the enzyme is a label, as in an enzymeimmunoassay, the quantity of enzyme present is related to the quantityof estradiol present in the test sample. Thus, the measurement offluorescence can be used to determine to the quantity of estradiolpresent in the test sample. The amount of steroid conjugate on the solidphase or in solution can be correlated to the concentration of estradiolin the test sample by means of a plot showing enzyme activity as afunction of estradiol concentration, typically referred to as a standardcurve. A standard curve can be prepared by performing the assay usingcalibrators such as those set forth in Table I, which will be discussedlater. Controls are used to verify that the standard curve is valid.When a sample having an unknown estradiol level is assayed, the measuredassay signal is compared to the standard curve, and the estradiol levelcorresponding to the measured signal is the estradiol level of thesample.

The specific binding member may be bound to the solid phase by physicalor chemical means, preferably by means of a covalent bond. The specificbinding member should be bound to the solid phase in such a way thatsubstantially none of the specific binding members detach during thesubsequent reactions and wash steps. Regardless of the specific bindingmember and the coupling method selected, the specific binding membermust be able to bind to estradiol and to the steroid conjugate afterbeing coupled to the solid phase.

A solid phase according to the present invention may be a mixture ofmicroparticles with binding members specific for estradiol chemically orphysically bound to the microparticles. Microparticles that can be usedin this invention are preferably made of polymeric material, and morepreferably include microparticles derived from polymers having styreneunits or polymers having acrylate units. The microparticles arepreferably substantially spherical and preferably have radii rangingfrom about 0.1 μm to about 0.25 inches. A preferred method forseparating these particles from the test sample involves capture of themicroparticle on a porous matrix, such as a glass fiber.

Other solid phases that can be used include a mixture of magnetizablemicroparticles having binding members specific for estradiol chemicallyor physically bound to the microparticles. Magnetizable microparticlesthat are useful in this invention preferably have ferric oxide orchromium oxide cores and a polymeric coating. Such coatings arepreferably made from homopolymers and copolymers having styrene units,homopolymers and copolymers having carboxylated styrene units, orhomopolymers and copolymers having acrylate or methacrylate units. Othersolid phases that are known to those skilled in the art include thewalls of wells of reaction trays, tubes, polymeric beads, nitrocellulosestrips, membranes, and the like. Natural, synthetic, and naturallyoccurring materials that are synthetically modified can be used as thematerial of the solid phase. Such materials include polysaccharides,e.g., cellulosic materials, such as, for example, paper and cellulosicderivatives, such as cellulose acetate and nitrocellulose; silica;inorganic materials, such as, for example, deactivated alumina,diatomaceous earth, MgSO₄, or other inorganic finely divided materialuniformly dispersed in a porous polymeric matrix, wherein the matrix maycomprise one or more polymers such as homopolymers and copolymers ofvinyl chloride, e.g., polyvinyl chloride, vinyl chloride-propylenecopolymer, and vinyl chloride-vinyl acetate copolymer; cloth, bothnaturally occurring (e.g., cotton) and synthetic (e.g., nylon); porousgels, such as silica gel, agarose, dextran, and gelatin; polymericfilms, such as polyacrylamide; and the like. In any case, the solidphase should have sufficient strength to maintain the desired physicalshape and should not interfere with the production of a detectablesignal. Strength can be provided by means of a support.

An alternative separation method is described in U.S. patent applicationSer. Nos. 150,278, abandoned, filed Jan. 20, 1988 and 375,029,abandoned, filed Jul. 7, 1989, both of which enjoy common ownership andboth of which are incorporated herein by reference. A publishedcounterpart of this reference is WO 92/21980, published Dec. 10, 1992,incorporated herein by reference. These references describe a methodinvolving ion capture separation, in which the specific binding membersused in the assay are chemically attached to both a first polyioniccompound and to a porous matrix having bound thereto a second polyioniccompound that binds to the first polyionic compound. A specific bindingpair is formed and separated from the reaction mixture by anelectrostatic interaction between the first and second polyioniccompounds. A specific binding member of the specific binding pair ispreferably covalently coupled to the first polyionic compound.

Preferably, the first polyionic compound is a polyanionic acid, such aspolyaspartic acid, heparin, carboxylmethyl amylose, polyglutamic acid,or polyacrylic acid, and the second polyionic compound is a cationicpolymer, such as a polymeric quaternary ammonium compound ("GafQuattum",GAF Corporation, Wayne, N.J., 07470), diethylaminoethyl-dextran (SigmaChemical Company, St. Louis, Mo.), water soluble cellulose derivatives,such as those having the trademarks "Celquat L-200" and "Celquat H-100"(National Starch & Chemical Corporation, Bridgewater, N.J. 08807), bothof which are polymeric quaternary compounds, or Merquat® 100 (CalgonCorporation). The porous matrix is treated with the cationic polymer toprovide a positive charge to the matrix. The cationic polymer is boundto the matrix by absorption, adsorption, or covalent or ionic coupling.The separation of the reaction products is effected by the electrostaticinteraction between the positively-charged matrix and thenegatively-charged polyanion complex.

A porous matrix suitable for use in this invention can be any suitableporous material. As used herein, "porous material" means a materialthrough which fluids can flow and can easily pass. Representativeexamples of materials suitable for the porous matrix include, but arenot limited to, olefin polymers, e.g., polypropylene, polyethylene,fluorinated olefin polymers, e.g., polytetrafluorethylene, fiberglass,cellulose, or nylon.

Preferred materials for the porous matrix include a porous fiberglassmaterial, such as a "Whatman 934-AH" filter paper, which has a nominalthickness of 0.33 mm, or the disposable IMx® cartridge and TestPack™(fiber matrix) devices (Abbott Laboratories, Abbott Park, Ill.; 60064).The thickness of such material is not critical, and will be a matter ofchoice, based upon the properties of the test sample or analyte beingassayed, such as the fluidity of the test sample.

As stated previously, an enzyme substrate can be converted into afluorescent compound, such as 4-methylumbelliferone, by an appropriateenzyme. The rate at which the fluorescent compound is formed is anindication of the quantity of enzyme present in the reaction mixture.When the enzyme is a label, the quantity of enzyme present is related tothe quantity of estradiol present in the test sample. Thus, themeasurement of fluorescence resulting from enzyme activity in an enzymeimmunoassay can be used to determine to the quantity of estradiolpresent in the test sample. Fluorescence can be measured by any methodknown to the art. For example, a fluorescence spectrometer can be used.The fluorescence spectrum can also be observed by means of a visualspectrometer or by a photograph with a spectrograph of highlight-gathering power.

In a preferred embodiment, the fluorescence can be detected by using anIMx® automated bench top analyzer (Abbott Laboratories, Abbott Park,Ill.). This analyzer contains an optical assembly comprising afluorometer that uses a mercury arc lamp as its light source. Thisinstrument is described by Fiore et al (Clin. Chem., 34/9:1726-1732,1988), incorporated herein by reference. The instrument utilizes IMx®disposable cartridges (commercially available from Abbott Laboratories,Abbott Park, Ill.), which contain a porous matrix to capturemicroparticles containing anti-estradiol antibodies that have beenexposed to a conjugate and the sample. The label used in the conjugateis preferably alkaline phosphatase. The estradiol in the sample binds toanti-estradiol antibodies on the microparticles. The microparticles areseparated from the reaction mixture, conjugate is added to themicroparticles, conjugate binds to available anti-estradiol antibodies,and the quantity of conjugate present on the microparticles isdetermined from the rate at which 4-methylumbelliferyl phosphate isconverted into 4-methylumbelliferone. The quantity of estradiol in thesample can be determined from a standard curve of rate of4-methylumbelliferone formation as a function of estradiolconcentration. The standard curve is also known as a calibration curve.

The standard curve that relates the rate of 4-methylumbelliferoneformation to estradiol concentration is generally prepared fromcalibrator solutions containing known estradiol concentrations.Preferably, six calibrators are used to obtain a calibration curve,though more or fewer calibrators can be used, depending on the accuracyand precision of the result desired. Preferably, the calibrators containincreasing amounts of estradiol. For example, Table I illustrates thecompositions of one set of calibrators, i.e. for IMx® Estradiol assay.Controls are generally used in conjunction with an assay to confirm thevalidity of a calibration curve or assay reagents. The formulation ofthe controls may be different from that of the calibrators, and theestradiol concentration of a given control may not be identical withthat of any one of the calibrators. For example, controls having anestradiol concentration of 150, 500, and 1125 pg/mL would be suitablecontrols for the calibrators in Table I. One skilled in the art would becapable of devising other calibrator and control formulations.

                  TABLE I                                                         ______________________________________                                        Calibrator Estradiol concentration (pg/mL)                                    ______________________________________                                        F          3000                                                               E          1500                                                               D          750                                                                C          250                                                                B          50                                                                 A          0                                                                  ______________________________________                                    

To maintain aseptic conditions throughout the procedure, it may bedesirable to add small quantity of an anti microbial agent to the systemwhich may include solvents, antibiotics and poisons.

The following examples are illustrative of the invention and are not tobe interpreted as limiting the scope of the invention, as defined in theclaims. All percentages are by grams (g) weight per 100 milliliters (mL)volume (w/v), unless otherwise indicated.

EXAMPLE 1 Estradiol Assay

Estradiol assays were performed on IMx® disposable cartridges by an IMx®instrument according to the following procedure. A serum sample (75 μL)was mixed with IMx® Estradiol Assay buffer (35 μL), IMx® Estradiolmicroparticles coated with anti-estradiol antibody (50 μL), and IMx®Buffer (90 μL) to form a reaction mixture. The reaction mixture wasincubated for 27.5 minutes at a temperature of 35° C.

IMx® Estradiol Assay buffer was composed of 5α-dihydrotestosterone (2μg/mL), 0.75% (w/v) saponin, 0.5 M glycine, 0.25 mM sodium citrate, and0.12% methyl isothiazolinone (w/v), all at pH 4.5. IMx® Estradiolmicroparticles coated with anti-estradiol antibody (0.005-0.02% solids,w/v) were suspended in IMx® Estradiol Assay buffer, which was composedof 0.1 M bis-(2-hydroxyethyl)iminotris(hydroxymethyl) methane(hereinafter "Bis-Tris"), 0.1 M sodium chloride, 13.6% sucrose (w/v),0.1% sodium azide (w/v), and 0.2 mg/mL normal rabbit IgG, all at pH 6.5.IMx® Buffer was composed of 0.3 M NaCl, 0.1 M tris(hydroxymethyl)aminomethane (hereinafter "Tris"), 0.1% sodium azide (w/v), all at pH 7.5.

One hundred seventy five microliters (175 μL) of the reaction mixturewere transferred to the fiber matrix of an IMx® disposable cartridge.The fiber matrix was located over an absorbent pad of the IMx®cartridge. The microparticles were captured by the fiber matrix, and thesolution was absorbed by the absorbent pad. The microparticles were thenwashed with IMx® Buffer. A steroid-alkaline phosphatase conjugate (60μL) was added to the matrix, incubated for 12 seconds at a temperatureof 35° C., and then washed again with IMx® Buffer. The conjugate (2-8μg/mL alkaline phosphatase) was in conjugate buffer composed of 0.1 MBis-Tris, 0.5 M sodium chloride, 1% casein (w/v), 1 mM magnesiumchloride, 0.1 mM zinc chloride, 0.1% azide (w/v), all at pH 6.5. Thepreparation of various steroid-alkaline phosphatase conjugates isdescribed in Example 4.

A 1.2 mM solution of 4-methylumbelliferone phosphate (65 μL) in 0.1 M2-amino-2-methyl-1-propanol buffer, at pH 9, was added to the matrix,and the rate of 4-methylumbelliferone formation was measured byfluorescence reflectance. The IMx® instrument measured fluorescence witha fluorometer that used a mercury arc lamp as its light source, asdescribed in Fiore et al., Clin. Chem., 34/9: 1726-1732, 1988, thecontents of which were previously incorporated by reference.

Materials needed for the assay, including IMx® Estradiol buffer, IMx®Estradiol anti-estradiol antibody coated microparticles, conjugate,methylumbelliferone phosphate substrate, IMx® Buffer, IMx® disposablecartridges, and IMx® instruments are available commercially from AbbottLaboratories, Abbott Park, Ill. and are described in U.S. Pat. No.5,342,760, European Application No. EP-A-288 793, and in Fiore et al.,Clin. Chem. 34/9: 1726-1732, 1988, all of which are incorporated hereinby reference.

EXAMPLE 2 Progesterone Assay

Progesterone assays were performed on IMx® disposable cartridges by anIMx® instrument according to a procedure similar to that used in theestradiol assay in Example 1, with the following modifications. A serumsample (50 μL) was mixed with progesterone sample buffer (80 μL),microparticles coated with anti-progesterone antibody (80 μL), and IMx®Buffer (40 μL). The reaction mixture was incubated for 20.8 minutes at atemperature of 35° C.

Progesterone sample buffer was composed of 2.25 M glycine,5α-dihydrotestosterone (0.5 μg/mL), 0.2% (w/v) saponin (EM Industries,Hawthorne, N.Y.), 0.1 M sodium chloride, and 0.1% methyl isothiazolinone(w/v, Rohm and Haas, Philadelphia, Pa.), all at pH 2.8. Themicroparticles (Seradyne, Indianapolis, Ind.) coated withanti-progesterone antibody (BiosPacific, Emeryville, Calif.) contained0.003% (w/v) solids suspended in a buffer that contained 0.5 Mmorpholinoethanesulphonic acid (MES), 0.1 M sodium chloride, 10% sucrose(w/v), 2% bovine serum albumin (w/v, Intergen, Purchase, N.Y.), 0.1mg/mL mouse IgG (50% (w/v) ammonium sulfate cut of mouse serum obtainedfrom Stellar Biosysytems, Columbia, Md.), and 0.2% sodium azide (w/v),all at pH 6.5

One hundred seventy five microliters (175 μL) of the reaction mixturewere transferred to the fiber matrix of an IMx® disposable cartridge.The microparticles were then washed with IMx® Buffer. A steroid-alkalinephosphatase conjugate (60 μL) was added to the matrix, the reaction wasincubated for 8 seconds at a temperature of 35° C., and then washedagain with IMx® Buffer. The conjugate (2-15 μg/mL alkaline phosphatase)was in a buffer composed of 50 mM Tris, 100 mM sodium chloride, 10 mMmagnesium chloride, 0.1 mM zinc chloride, 0.5% casein (w/v, SigmaChemical Company, St. Louis, Mo.), and 0.1% sodium azide (w/v), all atpH 7.4.

The addition of 4-methylumbelliferone phosphate and measurement of therate of 4-methylumbelliferone formation was carried out according to themethod described in Example 1.

EXAMPLE 3

Preparation of Antibody-Coated Microparticles

Microparticles coated with antibody were prepared as follows:

a. Murine anti-estradiol monoclonal antibodies (BiosPacific A3268;Biogenesis 2F-9, Sandown N.H.; Medix MBE 0107, San Carlos, Calif.) andthe anti-progesterone monoclonal antibody (BiosPacific 087) wereobtained from vendors. Anti-estradiol polyclonal antibodies (BiosPacificB4996 and Medix M17794) were also obtained from a vendor.

b. Rabbit anti-estradiol polyclonal antibodies were prepared byinjecting rabbits with estradiol 6-(O-carboxymethyl)oxime (SigmaChemical Company, St. Louis, Mo. and Steraloids, Wilton, N.H.) coupledto bovine serum albumin in Freund's complete adjuvant and boosting withFreund's incomplete adjuvant.

c. Latex microparticles (sufficient for a final reaction concentrationof 1% solids, w/v) were mixed with 50 mM MES buffer (pH 4.5) andantibody at 1 mg/mL. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDAC) was added to give a final concentration of 0.5 mg/mL. After themixture was incubated for 30 minutes at room temperature, themicroparticles were washed three times with 0.1 M Tris (pH 7.4)containing 0.1% "Tween 20" surfactant (v/v) and 0.1 M sodium chloride.The microparticles were then resuspended in microparticle storagebuffer. The anti-estradiol antibody-coated microparticle storage bufferis described in Example 1, and the anti-progesterone antibody-coatedmicroparticle storage buffer is described in Example 2. Assaysformulated with these microparticles are described in Example 5.

EXAMPLE 4

Preparation of Steroid-Alkaline Phosphatase Conjugates

Steroid alkaline-phosphatase conjugates were prepared as follows:

a. Ten micrograms (10 μg) of steroid derivative used for a particularconjugation and-10 μg of N-hydroxysuccinimide were dissolved in 100microliters (100 μL) of dimethylformamide (anhydrous). Forty micrograms(40 μg) of EDAC was added to the solution. The steroids used to producevarious conjugates utilizing estradiol analogues and progesteroneanalogues with calf intestinal alkaline phosphatase are listed in TableII.

b. An aliquot of the mixture in step a was added to alkaline phosphatase(available from Boehringer Mannheim, Germany) in carbonate buffer at pH8 so that the molar ratio of steroid to enzyme was 10:1 (20:1 fortestosterone). The resulting steroid-alkaline phosphatase conjugate wasseparated from unbound steroid and other low molecular weight materialson a G25 Sephadex column. Aliquots of the separated conjugate weredrawn, and these aliquots were diluted to a concentration ofapproximately 1 to 20 μg/mL in either the estradiol or progesteroneconjugate buffers. Example 1 describes the conjugate buffer used inestradiol assays and the assay employing the IMx® instrument. Example 2describes the conjugate buffer used in progesterone assays and the assayemploying the IMx® instrument.

                  TABLE II                                                        ______________________________________                                                 Derivative used for                                                  Steroid  conjugation     Symbol  Catalog                                                                             Vendor                                 ______________________________________                                                 conjugation                                                          Estradiol                                                                              1,3,5,[10]-Estratrien-3,17β3                                                             E2      K2126  A.sup.2                                        diol-6-one 6-CMO.sup.1                                               Estrone  1,3,5,[10]-Estratrien-3-ol-6,                                                                 E1      E2055  B.sup.3                                        17-dione 6-CMO                                                       Estriol  1,3,5,[10]-Estratrien-3,16α,                                                            E3      K2501 A                                               17β-triol-6-one 6-CMO                                           Ethynyl  1,3,5,[10]-Estratrien-17α-                                                              EE2     E1582 B                                      estradiol                                                                              ethynyl-3,17β-diol-6-one                                                 6-CMO                                                                17α-Estradiol                                                                    1,3,5,[10]-Estratrien-3,17α-                                                            17αE2                                                                           E1335 B                                               diol-6-one 6-CMO                                                     20-Hydroxy                                                                             4-Pregnen-20α-ol-3-one                                                                  20αP                                                                            Q3605 B                                      progesterone                                                                           3-CMO                                                                17-Hydroxy                                                                             4-Pregnen-17α-ol-3,20-                                                                  17αP                                                                            Q3375 B                                      progesterone                                                                           dione 3-CMO                                                          Testosterone                                                                           4-Androsten-17β-ol 3,                                                                    T2      2012-1                                                                               C.sup.4                                        19-dione 19-CMO                                                      ______________________________________                                         .sup.1 Sigma Chemical Company, St. Louis, MO                                  .sup.2 Steraloids, Wilton, NH                                                 .sup.3 Research Plus, Bayonne, NJ                                             .sup.4 CMO = [Ocarboxymethyl]oxime                                       

EXAMPLE 5

Anti-estradiol Antibody/Steroid Conjugate Matrix

Several anti-estradiol antibodies, including both the murine monoclonalantibodies and rabbit polyclonal antibodies, were tested for rate ratiovalue with a wide variety of steroid alkaline phosphatase conjugates inthe assay format described in Example 1. In addition, ananti-progesterone antibody was tested with three steroid alkalinephosphatase conjugates in the assay format described in Example 2. Theresults are set forth in Table III.

The dose response is assessed by measuring rate ratio values. As usedherein, "rate ratio value" means the quotient obtained by dividing thevalue of the assay signal observed at a particular analyte concentration(in this example, Table III, Calibrator F) by the value of the assaysignal observed with no analyte (in this example, Table III, CalibratorA). The Abbott IMx® instrument measures the assay signal in units offluorescent counts per second per second (cps/s). The rate ratio valueis a dimensionless number and normalizes the data from different assays,thereby allowing for direct comparison of different assays or assayformats. For convenience, the rate ratio value was calculated from thesignals from Calibrator F and from Calibrator A. In the estradiol assay,Calibrator F is at a concentration of 3000 pg/mL estradiol, and in theprogesterone assay, Calibrator F is at a concentration of 40 ng/mLprogesterone. Calibrator A contains no analyte.

A representative sampling of rate ratio values for the CalibratorF/Calibrator A rates from a screen of over 700 differentantibody/steroid conjugate combinations is shown in Table III. Eightsteroid conjugates and a representative number of murine monoclonal andrabbit polyclonal antibodies with exceptionally low rate ratio valuesare illustrated. As an example, for the polyclonal antibody Rabbit 438,the lowest rate ratio value for the eight steroid conjugates tested wasfor the ethynyl estradiol (EE2) conjugate, where the rate ratio valuewas 0.20. For all of the 700 antibody/steroid conjugate combinationstested, neither the estradiol nor estrone conjugate generated the bestdose response in the estradiol assay. In this example, the estradiol(E2) and estrone (E1) embodiments are comparative examples. They do notconstitute analogues for the estradiol assay.

                                      TABLE III.sup.1                             __________________________________________________________________________             E1  E2 E3  E E2                                                                             17αE2                                                                        2OαP                                                                        17αP                                                                        T2                                        __________________________________________________________________________    Anti-estradiol                                                                monoclonal                                                                    antibodies                                                                    BiosPacific A3268                                                                      0.20                                                                              0.25                                                                             0.14                                                                              0.23                                                                             0.45 0.94                                                                              nd.sup.2                                                                          nd                                        Biogenesis 2F9                                                                         0.30                                                                              0.23                                                                             0.18                                                                              0.25                                                                             0.85 1.02                                                                              nd  nd                                        Medix MBE 0107                                                                         0.57                                                                              0.69                                                                             0.50                                                                              0.48                                                                             0.26 0.38                                                                              1.02                                                                              0.77                                      Anti-estradiol                                                                polyclonal                                                                    antibodies                                                                    Medix M17794                                                                           0.26                                                                              0.37                                                                             0.18                                                                              0.32                                                                             0.31 1.00                                                                              0.89                                                                              0.83                                      Rabbit 438.sup.3                                                                       0.38                                                                              0.41                                                                             0.37                                                                              0.20                                                                             0.37 1.07                                                                              0.89                                                                              0.57                                      Rabbit 9959                                                                            0.40                                                                              0.50                                                                             0.39                                                                              0.30                                                                             0.47 0.84                                                                              1.09                                                                              0.55                                      Rabbit 9969                                                                            0.45                                                                              0.53                                                                             0.44                                                                              0.31                                                                             0.35 0.92                                                                              0.81                                                                              0.94                                      Rabbit 9965                                                                            0.21                                                                              0.27                                                                             0.16                                                                              0.17                                                                             0.13 0.85                                                                              nd  nd                                        Rabbit 581                                                                             0.23                                                                              0.24                                                                             0.18                                                                              0.14                                                                             0.09 0.97                                                                              0.86                                                                              0.78                                      Rabbit 583.sup.4                                                                       0.09                                                                              0.16                                                                             0.09                                                                              0.16                                                                             0.03 0.84                                                                              0.87                                                                              0.43                                      Rabbit 2522                                                                            0.73                                                                              0.69                                                                             0.67                                                                              0.62                                                                             0.61 0.22                                                                              0.99                                                                              0.39                                      BiosPacific B4996                                                                      0.62                                                                              0.72                                                                             0.62                                                                              0.50                                                                             0.36 0.85                                                                              0.32                                                                              0.49                                      Rabbit 2424                                                                            0.75                                                                              0.67                                                                             0.72                                                                              0.67                                                                             0.67 0.78                                                                              0.98                                                                              0.35                                      Anti-progesterone                                                             monoclonal                                                                    antibody                                                                      BiosPacific 087                                                                        0.15                                                                              nd nd  nd nd   nd  0.24                                                                              0.08                                      __________________________________________________________________________     .sup.1 See Table II for complete name of chemicals denoted by symbols.        .sup.2 not determined                                                         .sup.3 Assignee's rabbits are designated in the following manner: Rabbit      ABCD, where A, B, C, and D are numerals.                                      .sup.4 Antibody used in IMx ® Estradiol assay.                       

EXAMPLE 6

Dose Response Curves for Polyclonal Rabbit 583 Antibodies withConjugates of Estradiol and Estradiol Analogues

The pattern of the rate ratio values for a given steroid assay is uniquefor each antibody/steroid conjugate combination used, as illustrated inFIGS. 1-6. The rate ratio value is the ratio of the assay signalobserved at a particular analyte concentration to the assay signalobserved when no analyte is present. The rate ratio value can bemeasured at any desired analyte concentration. In this example,estradiol calibrators B, C, D, E, and F (from Table I) were used todetermine values of rate ratio over the clinically useful range forestradiol concentration. The values of rate ratio for a givenantibody/steroid conjugate combination are plotted as a function of theanalyte concentration to produce a dose response curve. The doseresponse curves of selected anti-estradiol antibody/conjugatecombinations were assessed over the dynamic range of the IMx® Estradiolassay (0-3000 pg/mL). FIG. 1 demonstrates the different dose responsecurves observed for the anti-estradiol polyclonal antibody from Rabbit583 and various estrogen conjugates.

It can be seen that low values of rate ratio for any particularcalibrator (e.g., Calibrator F), are associated with steep dose responsecurves. As used herein, the expression "steep dose response curve" meansthat a small change in concentration at low concentrations of analyte(i.e., in the abscissa of the curve) results in a large change in therate ratio value (i.e., in the ordinate of the curve). To furtherclarify this point, in FIG. 1 the dose response curve for 17αE2 issteeper than the dose response curves for E1 and E3, which curves aresteeper than the dose response curves for E2 and EE2. For polyclonalantibody Rabbit 583, the 17αE2 conjugate produced the steepest doseresponse curve, and similarly the lowest Calibrator F/Calibrator A rateratio value (0.03). See Table III and FIG. 1. The relative steepness ofdose response curves is inversely related to the magnitude of the rateratio value for Calibrator F (see Table III and Example 5).

A steeper dose response curve in the region of low concentration ofanalyte makes it possible to obtain a more accurate estimation ofanalyte concentration in that region. Accordingly, steeper dose responsecurves for a particular antibody/steroid combination in the region oflow concentration of analyte indicate that the combination will providean immunoassay that can more accurately measure relatively low steroidconcentrations, and, consequently, provide a more clinically relevantassay. The accurate measurement of small differences in concentration atlow steroid concentrations is considered one of the primary clinicalutilities of steroid assays used to diagnose fertility.

EXAMPLE 7

Dose Response Curves for Polyclonal Rabbit 2522 Antibodies withConjugates of Steroid Analogues

FIG. 2 illustrates the different dose response curves observed for theanti-estradiol polyclonal antibody from Rabbit 2522 and conjugates ofvarious steroid analogues. For this antibody, the lowest rate ratiovalue was observed with conjugate haptens other than those containing anaromatic A ring. The dose response curve of progesterone analogue 17αPis steeper than the dose response curve of testosterone T2, which issteeper than the dose response curves of estrogen 17αE2, EE2, and E2.

EXAMPLE 8

Dose Response Curves for Polyclonal Rabbit 438 Antibodies withConjugates of Estradiol and Estradiol Analogues

FIG. 3 illustrates the different dose response curves observed for theanti-estradiol polyclonal antibody from Rabbit 438 and either conjugateof estradiol or conjugates of various estradiol analogues. For thisantibody, the steepest dose response curve was observed with an ethynylestradiol conjugate hapten (EE2).

EXAMPLE 9

Dose Response Curves for Polyclonal Rabbit 581 Antibodies withConjugates of Estradiol and Estradiol Analogues

FIG. 4 illustrates the different dose response curves observed for theanti-estradiol polyclonal antibody from Rabbit 581 and either conjugatesof estradiol or conjugates of various estradiol analogues. For thisantibody, the steepest dose response curve was observed with a 17αestradiol conjugate hapten (17α E2).

EXAMPLE 10

Dose Response Curves for Monoclonal Murine A3268 Antibodies withConjugates of Estradiol and Estradiol Analogues

FIG. 5 illustrates the different dose response curves observed for theanti-estradiol murine monoclonal antibody A3268 from BiosPacific andeither conjugate of estradiol or conjugates of various estradiolanalogues. For this antibody, the steepest dose response curve wasobserved with an estriol conjugate hapten (E3).

EXAMPLE 11

Dose Response Curves for Monoclonal Murine 087 Antibodies withConjugates of Steroid Analogues

The dose response curves for anti-progesterone antibody/conjugatecombinations were assessed over the range of 0-40 ng/mL (progesteronecalibrators A through F). FIG. 6 illustrates the different dose responsecurves observed for the anti-progesterone murine monoclonal antibody 087from BiosPacific and conjugates of the steroid analogues 17α-hydroxyprogesterone (17αP) and testosterone (T2). For this antibody, asignificant improvement in dose response curve was observed with thetestosterone conjugate relative to the more structurally similarprogesterone analogue 17α-hydroxy progesterone. Calibrators A through Ffor the progesterone assays were as follows: Calibrator A=0 ng/mLprogesterone; Calibrator B=1.0 ng/mL progesterone; Calibrator C=4.0ng/mL progesterone; Calibrator D=10 ng/mL progesterone; Calibrator E=20ng/mL progesterone, and Calibrator F=40 ng/mL progesterone.

Various modifications and alterations of this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention, and it should be understood that thisinvention is not to be unduly limited to the illustrative embodimentsset forth herein.

What is claimed is:
 1. A method for determining the amount of estradiol in a test sample comprising the steps of:a. incubating a mixture of a test sample suspected of containing estradiol, a solid phase coupled to an antibody specific for estradiol, and a conjugate of an estradiol analogue to form estradiol/antibody complexes and conjugate/antibody complexes on said solid phase; b. separating said solid phase from said mixture; c. measuring the amount of label present in said mixture or in said solid phase; and d. determining the amount of estradiol in said sample from the amount of label, wherein said conjugate of said estradiol analogue has the formula: ##STR9## where R¹ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R² represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R³ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, and an alkynyl group, and an alkoxy group; R⁴ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, and an alkynyl group, and an alkoxy group; R⁵ represents an alkoxy group, or OH; L represents a label group; provided that the carbon atom at position 16 does not contain two substituents that are attached to the carbon atom at position 16 by an oxygen atom, and further provided that the carbon atom at position 17 does not contain two substituents that are attached to the carbon atom at position 17 by an oxygen atom, wherein said estradiol analogue has the formula: ##STR10##
 2. The method of claim 1, wherein said label is selected from the group consisting of catalysts, fluorescent compounds, chemiluminescent compounds, and enzymes.
 3. A method for determining the amount of estradiol in a test sample comprising the steps of:a. incubating a mixture of a test sample suspected of containing estradiol, a solid phase coupled to an antibody specific for estradiol, and a conjugate of an estradiol analogue to form estradiol/antibody complexes and conjugate/antibody complexes on said solid phase; b. separating said solid phase from said mixture; c. measuring the amount of label present in said mixture or in said solid phase; and d. determining the amount of estradiol in said sample from the amount of label, wherein said conjugate of said estradiol analogue has the formula: ##STR11## where R¹ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R² represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group; and an alkoxy group; R³ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, and an alkynyl group, and an alkoxy group; R⁴ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁵ represents an alkoxy group, or OH; L represents a label group; provided that the carbon atom at position 16 does not contain two substituents that are attached to the carbon atom at position 16 by an oxygen atom, and further provided that the carbon atom at position 17 does not contain two substituents that are attached to the carbon atom at position 17 by an oxygen atom, wherein said estradiol analogue has the formula: ##STR12##
 4. The method of claim 3, wherein said label is selected from the group consisting of catalysts, fluorescent compounds, chemiluminescent compounds, and enzymes.
 5. A method for determining the amount of estradiol in a test sample comprising the steps of:a. incubating a mixture of a test sample suspected of containing estradiol, a solid phase coupled to an antibody specific for estradiol, and a conjugate of an estradiol analogue to form estradiol/antibody complexes and conjugate/antibody complexes on said solid phase; b. separating said solid phase from said mixture; c. measuring the amount of label present in said mixture or in said solid phase; and d. determining the amount of estradiol in said sample from the amount of label, wherein said conjugate of said estradiol analogue has the formula: ##STR13## where R¹ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R² represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R³ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, and an alkynyl group, and an alkoxy group; R⁴ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁵ represents an alkoxy group, or OH; L represents a label group; provided that the carbon atom at position 16 does not contain two substituents that are attached to the carbon atom at position 16 by an oxygen atom, and further provided that the carbon atom at position 17 does not contain two substituents that are attached to the carbon atom at position 17 by an oxygen atom, wherein said estradiol analogue has the formula: ##STR14##
 6. The method of claim 5, wherein said label is selected from the group consisting of catalysts, fluorescent compounds, chemiluminescent compounds, and enzymes.
 7. A kit for performing a competitive immunoassay for estradiol comprising:a solid phase coupled to an antibody specific for estradiol, and a conjugate of an estradiol analogue, wherein said conjugate of said estradiol analogue has the formula: ##STR15## where R¹ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R² represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R³ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁴ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁵ represents an alkoxy group, or OH; L represents a label group; provided that the carbon atom at position 16 does not contain two substituents that are attached to the carbon atom at position 16 by an oxygen atom, and further provided that the carbon atom at position 17 does not contain two substituents that are attached to the carbon atom at position 17 by an oxygen atom, wherein said estradiol analogue has the formula: ##STR16##
 8. The kit of claim 1, wherein said label is selected from the group consisting of catalysts, fluorescent compounds, chemiluminescent compounds, and enzymes.
 9. A kit for performing a competitive immunoassay for estradiol comprising:a solid phase coupled to an antibody specific for estradiol, and a conjugate of an estradiol analogue, wherein said conjugate of said estradiol analogue has the formula: ##STR17## where R¹ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R² represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R³ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁴ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁵ represents an alkoxy group, or OH; L represents a label group; provided that the carbon atom at position 16 does not contain two substituents that are attached to the carbon atom at position 16 by an oxygen atom, and further provided that the carbon atom at position 17 does not contain two substituents that are attached to the carbon atom at position 17 by an oxygen atom,, wherein said estradiol analogue has the formula: ##STR18##
 10. The kit of claim 9, wherein said label is selected from the group consisting of catalysts, fluorescent compounds, chemiluminescent compounds, and enzymes.
 11. A kit for performing a competitive immunoassay for estradiol comprising:a solid phase coupled to an antibody specific for estradiol, and a conjugate of an estradiol analogue, wherein said conjugate of said estradiol analogue has the formula: ##STR19## where R¹ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R² represents a member selected from the group consisting of OH; H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R³ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁴ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁵ represents an alkoxy group, or OH; L represents a label group; provided that the carbon atom at position 16 does not contain two substituents that are attached to the carbon atom at position 16 by an oxygen atom, and further provided that the carbon atom at position 17 does not contain two substituents that are attached to the carbon atom at position 17 by an oxygen atom, wherein said estradiol analogue has the formula: ##STR20##
 12. The kit of claim 11, wherein said label is selected from the group consisting of catalysts, fluorescent compounds, chemiluminescent compounds, and enzymes.
 13. A method for determining the amount of progesterone in a test sample comprising the steps of:a. incubating a mixture of a test sample suspected of containing progesterone, a solid phase coupled to an antibody specific for progesterone, and a conjugate of an progesterone analogue to form progesterone/antibody complexes and conjugate/antibody complexes on said solid phase; b. separating said solid phase from said mixture; c. measuring the amount of label present in said mixture or in said solid phase; and d. determining the amount of progesterone in said sample from the amount of label, wherein said conjugate of said progesterone analogue has formula: ##STR21## where R⁶ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁷ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁸ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group; and an alkoxy group; R⁹ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; L represents a label group; provided that the carbon atom at position 16 does not contain two substituents that are attached to the carbon atom at position 16 by an oxygen atom, and further provided that the carbon atom at position 17 does not contain two substituents that are attached to the carbon atom at position 17 by an oxygen atom, wherein said progesterone analogue has the formula: ##STR22##
 14. The method of claim 13, wherein said label is selected from the group consisting of catalysts, fluorescent compounds, chemiluminescent compounds, and enzymes.
 15. A method for determining the amount of progesterone in a test sample comprising the steps of:a. incubating a mixture of a test sample suspected of containing progesterone, a solid phase coupled to an antibody specific for progesterone, and a conjugate of an progesterone analogue to form progesterone/antibody complexes and conjugate/antibody complexes on said solid phase; b. separating said solid phase from said mixture; c. measuring the amount of label present in said mixture or in said solid phase; and d. determining the amount of progesterone in said sample from the amount of label, wherein said conjugate of said progesterone analogue has formula: ##STR23## where R⁶ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁷ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁸ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁹ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; L represents a label group; provided that the carbon atom at position 16 does not contain two substituents that are attached to the carbon atom at position 16 by an oxygen atom, and further provided that the carbon atom at position 17 does not contain two substituents that are attached to the carbon atom at position 17 by an oxygen atom, wherein said progesterone analogue has the formula: ##STR24##
 16. The method of claim 15, wherein said label is selected from the group consisting of catalysts, fluorescent compounds, chemiluminescent compounds, and enzymes.
 17. A method for determining the amount of progesterone in a test sample comprising the steps of:a. incubating a mixture of a test sample suspected of containing progesterone, a solid phase coupled to an antibody specific for progesterone, and a conjugate of an progesterone analogue to form progesterone/antibody complexes and conjugate/antibody complexes on said solid phase; b. separating said solid phase from said mixture; c. measuring the amount of label present in said mixture or in said solid phase; and d. determining the amount of progesterone in said sample from the amount of label, wherein said conjugate of said progesterone analogue has formula: ##STR25## where R⁶ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁷ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁸ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁹ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; L represents a label group; provided that the carbon atom at position 16 does not contain two substituents that are attached to the carbon atom at position 16 by an oxygen atom, and further provided that the carbon atom at position 17 does not contain two substituents that are attached to the carbon atom at position 17 by an oxygen atom, wherein said progesterone analogue has the formula: ##STR26##
 18. The method of claim 17, wherein said label is selected from the group consisting of catalysts, fluorescent compounds, chemiluminescent compounds, and enzymes.
 19. A kit for performing a competitive immunoassay for progesterone comprising:a solid phase coupled to an antibody specific for progesterone, and a conjugate of a progesterone analogue, wherein said conjugate of said progesterone analogue has formula: ##STR27## where R⁶ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁷ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁸ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁹ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; L represents a label group; provided that the carbon atom at position 16 does not contain two substituents that are attached to the carbon atom at position 16 by an oxygen atom, and further provided that the carbon atom at position 17 does not contain two substituents that are attached to the carbon atom at position 17 by an oxygen atom, wherein said progesterone analogue has the formula: ##STR28##
 20. The kit of claim 19, wherein said label is selected from the group consisting of catalysts, fluorescent compounds, chemiluminescent compounds, and enzymes.
 21. A kit for performing a competitive immunoassay for progesterone comprising:a solid phase coupled to an antibody specific for progesterone, and a conjugate of a progesterone analogue, wherein said conjugate of said progesterone analogue has formula: ##STR29## where R⁶ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁷ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁸ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁹ represents a member selected from the group consisting of OH, H,. an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; L represents a label group; provided that the carbon atom at position 16 does not contain two substituents that are attached to the carbon atom at position 16 by an oxygen atom, and further provided that the carbon atom at position 17 does not contain two substituents that are attached to the carbon atom at position 17 by an oxygen atom, wherein said progesterone analogue has the formula: ##STR30##
 22. The kit of claim 21, wherein said label is selected from the group consisting of catalysts, fluorescent compounds, chemiluminescent compounds, and enzymes.
 23. A kit for performing a competitive immunoassay for progesterone comprising:a solid phase coupled to an antibody specific for progesterone, and a conjugate of a progesterone analogue, wherein said conjugate of said progesterone analogue has formula: ##STR31## where R⁶ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁷ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁸ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; R⁹ represents a member selected from the group consisting of OH, H, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group; L represents a label group; provided that the carbon atom at position 16 does not contain two substituents that are attached to the carbon atom at position 16 by an oxygen atom, and further provided that the carbon atom at position 17 does not contain two substituents that are attached to the carbon atom at position 17 by an oxygen atom, wherein said progesterone analogue has the formula: ##STR32##
 24. The kit of claim 23, wherein said label is selected from the group consisting of catalysts, fluorescent compounds, chemiluminescent compounds, and enzymes. 